Unpaired 2-tailed T-Test was performed on three end time points of MSD values.
P-values are presented from each test.

Figure 1c.
HeLa KRAS4b vs. HRAS
Time point	P-value		Significance at 95%
0.18		0.0183		Yes
0.19		0.0187		Yes
0.20		0.0189		Yes

HeLa KRAS4b vs. KRAS4a
Time point	P-value		Significance at 95%
0.18		0.0502		None
0.19		0.0517		None
0.20		0.0535		None

HeLa KRAS4b vs. NRAS
Time point	P-value		Significance at 95%
0.18		0.2441		None
0.19		0.2553		None
0.20		0.2610		None

Figure 1e.
MEF KRAS4b vs. HRAS
Time point	P-value		Significance at 95%
0.18		<0.0001		Yes
0.19		<0.0001		Yes
0.20		<0.0001		Yes

MEF KRAS4b vs. KRAS4a
Time point	P-value		Significance at 95%
0.18		0.0009		Yes
0.19		0.0009		Yes
0.20		0.0009		Yes

MEF KRAS4b vs. NRAS
Time point	P-value		Significance at 95%
0.18		0.0004		Yes
0.19		0.0004		Yes
0.20		0.0004		Yes

Figure 1g.
HeLa KRAS4b vs. KRAS4b HVR
Time point	P-value		Significance at 95%
0.18		0.0013		Yes
0.19		0.0014		Yes
0.20		0.0015		Yes

Figure 2c.
HeLa KRAS4b WT vs. KRAS4b ESR
Time point	P-value		Significance at 95%
0.18		0.1275		None
0.19		0.1303		None
0.20		0.1349		None

HeLa KRAS4b WT vs. KRAS4b GNK
Time point	P-value		Significance at 95%
0.18		0.0125		Yes
0.19		0.0153		Yes
0.20		0.0221		Yes

HeLa KRAS4b WT vs. KRAS4b HEK
Time point	P-value		Significance at 95%
0.18		0.7494		None
0.19		0.7893		None
0.20		0.8591		None

Figure 2d.
Diffusion coefficients and occupancies (three-component diffusion model) obtained from HMM analysis are not significantly different (at 95%) between G-Domain mutants.

Figure 3c.
Fast Diffusion Co-efficient	P-value		Significance at 95%
4BHVR-5Ea vs. 4bHVR-5A 		0.0133		Yes
4BHVR-5Ea vs. 4bHVR-3A		0.0087		Yes
4BHVR-5Ea vs. 4bHVR		0.0002		Yes
4BHVR-3A  vs. 4bHVR		0.0039		Yes

Figure 3e.
Residence time			P-value		Significance at 95%
4BHVR-5Ea vs. 4bHVR-5A 		0.0005		Yes
4BHVR-5Ea vs. 4bHVR-3A		0.0072		Yes
4BHVR-5Ea vs. 4bHVR		0.001		Yes

Figure 5a.
4B-WT vs. 4B-Y40C-Q61R 
Time point	P-value		Significance at 95%
0.18		0.0498		None
0.19		0.0545		None
0.20		0.0647		None

4B-WT vs. 4B-Y40C
Time point	P-value		Significance at 95%
0.18		0.0460		Yes
0.19		0.0522		None
0.20		0.0562		None

4B-Q61R vs. 4B-Y40C-Q61R
Time point	P-value		Significance at 95%
0.18		0.0222		Yes
0.19		0.0210		Yes
0.20		0.0204		Yes

Figure 5d.
Diffusion coefficients (three-component diffusion model) obtained from HMM analysis are not significantly different (at 95%) between Q61R and Y40C mutants. However, occupancy of fast and intermediate diffusing species is significantly (at 95%) different between Q61R/WT and Y40C/Q61R-Y40C variants. P-values are as follows.

4B-WT vs. 4B-Y40C	
Occupancy	P-value		Significance at 95%
Fast  		0.0254		Yes
Intermediate	0.067		No

4B-Q61R vs. 4B-Y40C
Occupancy	P-value		Significance at 95%
Fast  		0.009		Yes
Intermediate	0.010		Yes

4B-Q61R vs. 4B-Y40C-Q61R
Occupancy	P-value		Significance at 95%
Fast  		0.026		Yes
Intermediate	0.040		Yes

4B-Q61R vs. 4B-WT
Occupancy	P-value		Significance at 95%
Fast  		0.075		No
Intermediate	0.023		Yes


Figure 6a.
MEF KRAS4B-Serum Complete vs. Serum depleted
Time point	P-value		Significance at 95%
0.18		0.0036		Yes
0.19		0.0017		Yes
0.20		0.0004		Yes

MEF KRAS4B Serum depleted vs. Serum + 15min
Time point	P-value		Significance at 95%
0.18		0.0036		Yes
0.19		0.0162		Yes
0.20		0.0203		Yes

MEF KRAS4B Serum depleted vs. Serum + 60min
Time point	P-value		Significance at 95%
0.18		0.0256		Yes
0.19		0.0347		Yes
0.20		0.0410		Yes

Figure 6c.
Occupancy of fast diffusing species	P-value		Significance at 95%
Serum Complete vs. Serum depleted :	0.0053		Yes
Serum depleted vs. Serum + 15 min :	0.0531		None
Serum depleted vs. Serum + 60 min :	0.0029		Yes

Occupancy of intermediate and slow diffusing species are not significantly different (at 95%) between serum conditions. 

Diffusion coefficients are not significantly different (at 95%) between serum conditions.